Methylmercury Promotes Oxidative Stress and Activation of Matrix Metalloproteinases: Cardiovascular Implications

Abstract

Preclinical and clinical studies worldwide have shown an association between methylmercury (MeHg) poisoning and the risk of developing cardiovascular diseases such as arrhythmias, arterial hypertension, atherosclerosis and myocardial infarction. One of the hypotheses raised for MeHg-induced toxicity is associated with redox imbalance, which promotes oxidative stress by increasing reactive oxygen species (ROS) and reducing the activity of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx). In addition, oxidative stress and organomercurial compounds are capable of activating MMPs. MMP-2 and MMP-9 participate in pathophysiological processes associated with cardiovascular remodeling. A positive correlation between mercury exposure and increased plasma activity of MMP-2 and circulating MMP-9 has been demonstrated, suggesting a possible mechanism that could increase susceptibility to cardiovascular diseases.

1. Introduction

Mercury (Hg), an environmental pollutant from natural and anthropogenic sources, is converted into methylmercury (MeHg), a more toxic organic form capable of bioaccumulating in food webs [1, 2]. Humans are exposed to MeHg through the consumption of contaminated fish, especially predatory species, which through trophic magnification, accumulate higher amounts of the metal [3, 4]. The effects of human poisoning by MeHg took worldwide repercussions after the contamination of the Minamata Basin in Japan (1956). Years later, people who consumed fish from this region developed a “Minamata disease” syndrome, mainly affecting the nervous system [5].

Human exposure to MeHg can pose various health risks. The spectrum of adverse effects associated with MeHg poisoning will depend on the exposure time and magnitude of the dose [6, 7]. Life span also influences the damage caused by MeHg, and prenatal exposure can potentially cause irreversible damage to the developing central nervous system [8, 9]. The exposure that occurs during childhood and adulthood can also cause damage to the central nervous system. However, signs of toxicity appear months after the onset of exposure [10, 11].

In addition to affecting the Central Nervous System, MeHg compromises other systems [12, 13, 14]. For example, the repercussions of MeHg on the cardiovascular system have been investigated in recent decades [15, 16, 17]. A study of 1833 Finnish men aged 42–60 suggested a correlation between mercury accumulation in the body from high consumption of MeHg-contaminated non-fatty fish and the increased risk of acute myocardial infarction, coronary heart disease and other cardiovascular diseases. In this study, the researchers theorized that the effects of MeHg on the cardiovascular system could be associated with lipid peroxidation triggered by mercury [18].

One of the mechanisms involved in MeHg-induced cytotoxicity is the dysregulation of the redox state by increasing reactive species and suppressing the antioxidant system, triggering oxidative stress [19]. Furthermore, the membranes of cells and organelles are affected by the direct action of reactive species, which promote lipid peroxidation and generate changes in structure and permeability, which culminate in cell death [7, 20, 21]. In addition, MeHg promotes protein denaturation, enzyme inactivation, DNA damage and triggering epigenetic changes [22, 23].

Reactive species derived from oxygen and nitrogen have a crucial effect on the pathophysiology of cardiovascular diseases [24, 25]. These molecules activate matrix metalloproteinases (MMPs) [26, 27, 28]. The activation of MMPs can also occur directly by organomercurial compounds, which disrupt the interaction of a propeptide domain cysteine residue with catalytic zinc within the enzyme’s active site [29, 30]. MMPs are a family of zinc-dependent endopeptidases that control the synthesis and degradation of extracellular matrix (ECM) components and can also act directly on intracellular substrates on a small-time scale [31, 32].

The group of gelatinases (MMP-2 and MMP-9) is extensively investigated in cardiovascular diseases. The increase in proteolytic activity is closely related to the remodeling of the heart and vessels [33, 34, 35]. It has also been suggested the existence of a correlation between increased MMP-9 and MMP-2 activity and plasma mercury concentrations as a possible mechanism that could increase susceptibility to cardiovascular diseases, showing a possible causal relationship between mercurial exposure and increased susceptibility to cardiovascular diseases [36].

2. Forms of mercury

The chemical element Mercury (Hg) is a heavy metal whose symbol Hg derives from the Latin hydrargyrum, meaning liquid silver. It belongs to the transition metals in the sixth period and family II B of the periodic table. Under average temperature and pressure conditions, it presents a liquid physical state with silver coloration. Its fluidity at room temperature, and high thermal and electrical conductivity, make it an excellent conductive material. Furthermore, its uniform volumetric expansion over a wide temperature range and high density makes it an ideal element for manufacturing instruments for physical measurements, such as thermometers, barometers and electrical systems.

Among the natural sources emitting mercury in the environment are: volcanic eruptions, degassing of the Earth’s crust and mercury (HgS) mines [37, 38, 39]. The anthropogenic sources of mercury emission are multiple, emphasizing the chemical industry, with the burning of fossil fuels, the production of electronics, such as batteries, and the amalgamation of mercury used in dentistry and gold mining [40, 41, 42].

Mercury is widely distributed in nature, forming several compounds that are grouped into three groups: elemental mercury (Hg⁰), the inorganic species: cinnabar ore, mercury oxide, mercurous ion and mercuric ion (HeS, HgO, Hg2²⁺, Hg²⁺) respectively and the organic species, most common dimethylmercury ((CH₃)₂Hg) and methylmercury (CH₃Hg⁺). When mercury combines with carbon, it forms so-called organic or organomercurial compounds. The conversion of mercury to methylmercury (MeHg) occurs mainly in aquatic systems and can also be converted in soil and sediments in a smaller proportion [12, 43].

3. The mercury cycle in the environment and the methylation process

Understanding the dynamics of mercury in the environment is very important for understanding its actual impact on living things. The mercury cycle is characterized by the various routes that this compound can follow in the ambient carried by human or natural activity to the biosphere, transiting in different forms and states of oxidation, its distribution in the separate compartments that make up the environment: water, air and soil, produces extremely varied distribution patterns [44, 45].

In the atmosphere, the elemental mercury (Hg⁰), in the form of vapor, oxidizes, giving rise to the intermediate state of bivalent ionic mercury (Hg²⁺), which then complexes the molecule of ozone or chlorine, giving rise to substances (Hg^O and HgCl₂) less volatile and more soluble in water, which are easily solubilized in the water vapor of the clouds, which are subsequently deposited on the vegetation, soil and water. The Hg²⁺ also undergoes precipitation with rain, the most common form found in soil and surface water, and may undergo methylation or volatilize and return to the atmosphere [44, 45].

In the soil, the mercury deposited in the bivalent form can be complex to humic acids, clay mineral particles and mainly organic matter, conferring stability and high capacity to keep in the environment for long, even after eliminating the generating source. Furthermore, the mercury in the soil can be methylated or undergo sorption, converting into volatile forms that will return to the atmosphere or even dissolve in the aquatic environment, undergoing methylated and bioaccumulation in the food chain [44, 45, 46].

Inorganic mercury can be converted into elemental mercury in the aquatic ecosystem by microorganisms, humic and fulvic acids under specific physicochemical conditions. The major transformation undergone by inorganic mercury in the aquatic environment from the environmental point of view is its conversion into organomercurial compounds, among which methylmercury, the most toxic and bioavailable form, stands out. Surfactant bacteria interact with methylcobalamin, known as vitamin B12, transferring the methyl group to mercury, generating methylmercury [47, 48]. The synthesis rate of MeHg considers the composition of the species of bacteria, temperature, organic carbon, sulfur and dissolved oxygen for transfer of groups and acidity. Elemental mercury is little reactive in a natural aquatic environment, presenting a low possibility of oxidation, with practically zero contribution to the formation of MeHg [49, 50].

Methylmercury is stable in aquatic environments, forming several soluble complexes, thus favoring its dispersion. Methylmercury dissolved in water can be incorporated by plankton entering the food chain. Trophic magnification and bioaccumulation result in very high levels of MeHg in predatory fish, organisms at the top of the aquatic food chain, which are a significant source of exposure of wild animals and humans to MeHg [51, 52, 53] (Figure 1).

Figure 1.

Mercury cycle: Mercury can follow several routes in the different compartments that make up the environment, presenting various forms such as elemental mercury (Hg0), mercuric ion (Hg2+) and methylmercury (CH3Hg+), being released into the biosphere both by anthropogenic activity, burning of fossil fuel, and natural sources such as volcanic eruptions. In the atmosphere, Hg0, in the form of steam, can be converted into Hg2+ that is complex to other molecules, giving rise to substances that are associated with cloud water, precipitating with rain, depositing in water and soil, and may return to the atmosphere or undergo methylation by bacteria, and may be incorporated by plankton entering the food chain, suffers trophic magnification and bioaccumulation reaching organisms that are at the top of the food chain.

4. Toxic effects of methylmercury on human health

Throughout history, mercury has been used for various purposes, presenting a wide distribution in nature, resulting in intoxication in animal and human populations. However, the main form of human exposure to MeHg occurs from the diet, mainly from consuming contaminated fish [54, 55, 56, 57, 58].

Due to its liposolubility, MeHg ingested in food and rapidly absorbed by the gastrointestinal tract easily crosses the membranes of cells, the placenta and the blood-brain barrier [59, 60]. In addition, the systems in development are affected more severely due to the period of differentiation and cell migration, generating irreversible damage compared to adult organisms [61, 62, 63]. Therefore, populations whose diet is associated with the consumption of fish and marine mammals are at immediate risk of MeHg poisoning [54, 55, 56, 57, 58, 64].

MeHg is absorbed by the gastrointestinal tract and enters the bloodstream and bioaccumulates in different organs such as the liver, kidney, lung, brain and heart [65]. Because it is a metal with an electrophilic nature, MeHg reacts with nucleophiles such as selenol (SeH) and sulfhydryl (-SH) groups, forming stable complexes. In the intracellular environment, MeHg can form these complexes with proteins, as well as with GSH and the amino acid cysteine (Cys), which have these groups in their structures [66, 67, 68].

Conjugated MeHg circulates more easily in the blood and has greater permeability and distribution, as it now has a molecular structure similar to endogenous molecules [69]. Some studies have suggested that the complex formed by methylmercury-L-cysteine (MeHg-Cys) has a molecular structure identical to the amino acid methionine (Met), which crosses biomembranes through the L-type amino acid transporter (LAT1, LAT2 and LAT3), with However, the physiological function of this transporter still needs to be clarified, as well as its subcellular location, for a better understanding of MeHg toxicodynamics [70, 71, 72].

MeHg is rapidly taken into the intracellular medium, retained in subcellular regions, and slowly converted into inorganic mercury [73]. Available treatments for heavy metal poisoning use chelating agents such as 2,3-dimercaptopropanol (BAL), meso-2,3-dimercaptosuccinic (DMSA) and 2,3-dimercaptopropane-1-sulfonate (DMPS). However, these molecules are ineffective in MeHg detoxification, as they have a low therapeutic window, unable to remove the metal at the intracellular level. They can also cause the redistribution of the same in the body, often causing hepatotoxicity and nephrotoxicity, making it difficult to implement an appropriate treatment therapy in cases of MeHg intoxication [74, 75, 76].

The exact mechanism of cytotoxicity induced by MeHg is still not fully understood. However, many studies suggest that changes in the antioxidant system, calcium homeostasis and the glutamatergic system are involved in the toxic effects at the cellular level generated by MeHg [66].

In the Japanese city of Minamata in 1956, people who consumed fish and shellfish contaminated with MeHg, dumped by a chemical factory, developed a syndrome known as Minamata disease. People developed sensory disturbances, ataxia, dysarthria, visual field constriction, auditory changes and tremors. In addition, pregnant women in the region, who feed on species of this contaminated ecosystem, had children with extensive brain lesions [5, 11].

In Iraq, around 1971/1972, people were poisoned with MeHg by contamination of seeds with organomercurial compounds used as a fungicide. In addition, the consumption of homemade products prepared from these seeds caused the poisoning of many people, who presented as the most common symptom of paresthesia. In the most severe cases, the individuals exhibited ataxia, changes in vision, speech and hearing, followed by blindness, deafness and death [77, 78].

The two episodes of human poisoning by MeHg from consuming contaminated food served as bases to determine the effects of methylmercury. It was possible to establish that high doses of this metal in the body affect the nervous system and even lead to death. Furthermore, methylmercury could cross the transplacental barrier and cause irreversible damage to the fetus [11, 77, 79, 80].

However, human exposure to methylmercury generally occurs chronically and at low doses, and the spectrum of adverse effects and severity of damage largely depends on the magnitude of the exposure dose [81]. Children exposed to high amounts of methylmercury while still in the womb, as in the cases of Minamata and Iraq, were born with severe disabilities such as mental retardation, microcephaly, seizures, and blindness [82, 83, 84]. Exposure in utero to low doses of MeHg can generate effects that go unnoticed at birth. However, during development, the child presents neurocognitive deficits, such as difficulties in crawling, sitting, walking and learning [85].

Recently, epidemiological studies have been conducted to assess the human health risks of chronic exposure to low doses of MeHg. Two extensive studies that addressed the effects of MeHg on child development: the Seychelles Child Development Study (SCDS) and the Ilas Faroe Study, obtained divergent results. In the first SCDS, no adverse effects were found, while in the second Faroe Islands, a range of adverse effects was described, among them neuropsychological and neurophysiological impairment [86, 87, 88].

The effects caused by different doses of MeHg on the nervous system of adults and children have been extensively investigated over the last decades [89]. However, there is evidence that exposure to MeHg promotes changes in the cardiovascular system generating dysregulation of blood pressure, changes in heart rate and increased risk of developing coronary heart disease, atherosclerosis and acute myocardial infarction in adults [90, 91, 92].

5. Cardiovascular effects of MeHg and the role of oxidative stress, MMP-2 and MMP-9

MeHg is the major organic form of mercury involved in poisoning events in humans. It has relevant cardiovascular effects flagged in 2000 by the National Research Council on the Toxicological Effects of MeHg through a report that reinforced the need for more research. Clinical studies have observed associations between mercury levels and increased risk for myocardial infarction in Finland [18, 93, 94], Israel and eight European countries [95]. In addition, associations between mercury and arterial hypertension, vascular hypertrophy and arrhythmias were found in population studies in Wisconsin, the Brazilian Amazon region and among members of Faroese Whaling men [90, 91, 92].

Studies in cell and animal cultures have been performed to understand the cardiovascular toxicodynamic mechanisms of MeHg and demonstrated time-dependent effects. In acute exposure in rats, increased vascular relaxation was observed, with no changes in blood pressure [96]. On the other hand, in times of greater exposure, weight loss, mortality, hypertension, hypercholesterolemia, vascular hypertrophy, inflammation and atherosclerotic lesions and cardiac arrhythmias are observed [97, 98, 99, 100, 101]. Vascular findings have been associated with oxidative stress, inflammation and endothelial dysfunction [100, 101]. In fact, studies in endothelial cell culture have demonstrated that MeHg decreases cell viability [102, 103, 104] by the production of ROS and activation of phospholipases. These effects are prevented by the use of antioxidants [102, 104]. Subsequently, it was demonstrated that ROS production depends on the activation of NADPH oxidase and the decrease in antioxidant defense [105].

MeHg can bind directly to the tripeptide GSH by a sulfhydryl bond with the amino acid cysteine forming a complex (MeHg-GSH) that can be exported from the cell leading to depletion of its intracellular levels. However, this is not the only mechanism involved in reducing intracellular GSH levels. The greater interaction affinity of MeHg with selenocysteine residues present in GPx and the inhibition of its expression and activity alter the GSH-GSSG redox cycle, thus decreasing the GSH conversion rate [106, 107]. MeHg also can increase superoxide anion and hydrogen peroxide generation via NADPH oxidase and mitochondria, leading to intracellular GSH depletion. There is a dichotomous relationship between MeHg and GSH because this interaction favors MeHg excretion.

On the other hand, this interaction triggers cytotoxicity mechanisms. In addition, MeHg also decreases the activity of other antioxidant enzymes, such as SOD and catalase, contributing to increased ROS bioavailability [66, 97, 105, 108, 109]. In this way, MeHg induces oxidative stress that directly contributes to endothelial dysfunction.

Endothelial dysfunction is a key event in cardiovascular diseases, including hypertension, atherosclerosis and cardiomyopathies [110, 111, 112]. The leading cause and consequence of the loss of endothelial functionality is the decrease in nitric oxide (NO) production. NO can be produced by endothelial nitric oxide synthetase (eNOS), neuronal oxide synthetase (nNOS) and induced oxide synthetase (iNOS) that perform the conversion of the amino acid L-arginine and molecular oxygen into nitric oxide and L-citrulline. The reaction requires the presence of co-factors such as NADPH (nicotinamide adenine dinucleotide phosphate), BH4 (tetrahydrobiopterin), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) [113]. The main factor involved in the decrease in the bioavailability of NO is oxidative stress, resulting from the overproduction and inactivation of ROS such as superoxide (O₂⁻) leads to rapid chemical reaction with NO forming peroxynitrite (ONOO⁻), implying lower bioavailability of NO contributing to cardiovascular events [114]. Low MeHg concentrations induce hypertension in rats, which has been associated with oxidative stress and lower bioavailability of NO [97]. In addition to oxidative stress, MeHg generates S-mercuration, a post-translational modification in which mercury reacts with nucleophiles, such as the thiol group of proteins, leading to structural and functional alteration of molecules. MeHg has been described to cause S-mercuration of superoxide dismutase (SOD) and nNOS, thereby decreasing ROS inactivation and NO production [115, 116].

Organomercurial compounds such as MeHg can alter cardiovascular homeostasis also by modulating the activity of MMPs [29, 30]. MMPs control the content of the extracellular matrix and comprise a group of 28 proteins, 24 of which are found in humans. MMPs are classified according to their structure and primary degradation substrate. For example, MMP-2 and MMP-9 are present in the gelatinase group, the main MMPs studied in the cardiovascular system, mainly due to their relevant role and ease of evaluation with laboratory techniques. MMP-2 and MMP-9, similar to the other MMPs, have a signal peptide, a propeptide, a catalytic site containing a zinc atom and a hemopexin domain. However, they differ from the rest of the other enzymes by having three domains of fibronectin at the catalytic site, responsible for the high affinity for denatured collagen (gelatin) [117] (Figure 2).

Figure 2.

Proteolytic and non-proteolytic activation of MMP-2 and MMP-9. (A) the 72 kDa MMP-2 (pro-MMP-2) has in its structure a signal peptide (N), a propeptide, a catalytic domain containing zinc and three fibronectin repeats and a hemopexin domain. The sulfhydryl bond between a cysteine residue in the propeptide domain with zinc ensures catalytic inactivity. The active 72 kDa MMP-2 can undergo non-proteolytic activation by ROS and organomercurial compounds by breaking the sulfhydryl bond, maintaining the structure and molecular weight of 72 kDa. The isoform of active 64 kDa MMP-2 lacks the propeptide and can be formed by the proteolytic action of MMPs or serine proteases and by autolysis. (B) the 92 kDa MMP-9 (pro-MMP-9) has in its structure a signal peptide (N), a propeptide, a catalytic domain containing zinc and three fibronectin repeats, a collagen-binding domain type V and hemopexin domain. The proteolytic and non-proteolytic activation processes are similar to those previously described.

MMP-2 and MMP-9 expressed are secreted into the extracellular medium as the zymogen, called Pro-MMP-2 (72 kDa) and Pro-MMP-9 (92 kDa). Enzymatic inactivity is maintained by a sulfhydryl bond between a cysteine in the propeptide and zinc at the catalytic site [117]. The activation process requires breaking the sulfhydryl bond to expose the catalytic site, known as the “ cysteine switch” [118]. Briefly, the activation of gelatinases can occur by cleavage of the propeptide by other enzymes, leading to a decrease in the molecular weight of MMP-2 (64 kDa) and MMP-9 (83 kDa) or by non-proteolytic activation that can occur by disruption of the sulfhydryl bond by reactive species and mercurial compounds [26, 27, 28, 29, 30, 117]. The non-proteolytic activation maintains the molecular weight of the enzyme. However, the conformational change of the structure subsequently allows the propeptide’s auto-cleavage, resulting in a decrease in molecular weight (Figure 2). The enzymatic activity of MMP-2 and MMP-9 is modulated by tissue inhibitors of MMPs (TIMPs) and alpha2-macroglobulin. The four TIMPs described are not selective regarding the inhibition of MMPs, but MMP-2 and MMP-9 are inhibited mainly by TIMP-2 and TIMP-1, respectively [117].

MeHg can directly activate MMP-2 and MMP-9, increasing tissue proteolytic activity [29, 30]. In fact, increased activity of MMP-2 and MMP-9 was observed in population samples from the Amazon region of Brazil, which showed high plasma levels of mercury due to a diet rich in contaminated fish. The authors demonstrated a negative correlation between mercury levels and TIMP-1 and TIMP-2. The positive correlation between mercury levels and the ratio MMP-9/TIMP-1 and MMP-2/TIMP-2 leads to increased enzyme gelatinolytic activity [36]. Later studies demonstrated that polymorphisms in the MMP-2 and MMP-9 genes are related to changes in gelatinase activity and MMP-2/9/TIMPs ratio in mercurial intoxication [119, 120]. It has also been shown that MeHg can epigenetically alter MMP-9 by inducing changes in cell junctions [22]. Together, these studies demonstrated biological mechanisms that may be related to the cardiovascular toxicity of MeHg (Figure 3).

Figure 3.

Cardiovascular adverse events associated with MeHg poisoning: Humans become contaminated with MeHg from their diet, mainly by consuming contaminated fish. MeHg is ingested in food and rapidly absorbed by the gastrointestinal tract, crossing cell membranes, promoting cytotoxicity by altering the redox balance, generating an increase in reactive species and suppressing the antioxidant system, leading to oxidative stress. Oxidative stress and MeHg can activate MMP-2 and MMP-9. The higher proteolytic activities promote the remodeling and dysfunction of the heart and vessels, triggering adverse cardiovascular events.

The imbalance between the activity of MMPs and TIMPs is one of the main determinants of matrix composition and is related to increased cardiovascular risk [121, 122]. Studies have shown that increased MMP-2 activity in the heart leads to decreased cardiac systolic function due to the cleavage of sarcomere proteins, including troponin I, alpha-actinin, titin and light chain myosin [123, 124, 125, 126]. In these studies, intracellular activation of MMP-2 by ONOO⁻ was demonstrated to result in intracellular lysis of sarcomere proteins, resulting in severe cardiac damage. MMP-9 modulates inflammatory response by activating cytokines and chemokines, including TNFα, IL-1β, TGFβ and CXC-modified ligands [127, 128, 129]. In addition, MMP-2 and MMP-9 also activate profibrotic pathways in cardiovascular tissue [130, 131, 132, 133]. It is worth mentioning that mercury is also stored in the heart [134, 135, 136], which can lead to direct activation of MMPs and disruption of the thiol-zinc bond, which can generate adverse events similar to those observed in the model of ischemia and reperfusion of the heart.

Although we have pointed out evidence of the toxic cardiovascular effects of MeHg, some population studies have not found a correlation between mercury levels and cardiovascular changes [10, 91, 92, 137, 138]. Furthermore, despite the signaling that MeHg induces oxidative stress and that both directly activate MMPs, few studies demonstrate the participation of MMPs in cardiovascular toxicodynamic events associated with MeHg. However, this triple association resulted in functional alterations in other body systems. Decreased renal function by cytoskeleton disruption was associated with increased MMP-9 gene expression via oxidative stress induced by MeHg [22]. In another study, activation of MMP-9 and MMP-13 via MeHg-induced redox alteration led to altered embryonic development in zebrafish [139]. Neurodevelopmental alterations caused by MeHg have also been associated with gene alterations of MMP-1 and antioxidant enzymes [140].

In summary, we show evidence that MeHg can modulate directly or indirectly via oxidative stress the expression and activity of MMPs. As previously mentioned, small alterations in MMP activity can lead to serious cardiovascular alterations. Thus, the interaction of MeHg, oxidative stress and MMPs can be a point to be looked at more thoroughly, and this could be a mechanism involved in the cardiotoxicity of MeHg.

6. Conclusions

The studies discussed in this chapter have shown that MeHg poisoning, both in low doses and in high doses, in animal models and humans, is capable of causing adverse events in the cardiovascular system. The cytotoxicity induced by MeHg causes depletion of the antioxidant system, both of molecules such as glutathione, as well as of antioxidant enzymes (GPx, SOD and CAT) and increase of reactive species (superoxide and NO). Furthermore, MeHg and oxidative stress can alter the expression and activity of MMP-2 and MMP-9, suggesting a possible mechanism of susceptibility to cardiovascular diseases. However, well-defined epidemiological studies are needed to establish a cause-and-effect relationship between dietary MeHg poisoning associated with altered redox status and a correlation between the increased activity of MMP-2 and MMP-9 with increased developmental richness with cardiovascular diseases.

Acknowledgments

This work had partial financial support from Conselho Nacional de Desenvolvimento científico e Tecnológico (CNPq 427591/2018-0), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES) —Finance Code 001. The APC payment was supported by Pró-reitoria de Pesquisa e Pós-Graduação (PROPESP) from the Federal University of Pará (UFPA).

Conflict of interest

The authors declare no conflict of interest.